Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

OBJECTIVES:

1) To correlate the methylation status of the O⁶-methylguanine-DNA-methyltransferase (MGMT) promoter to its gene and protein expression levels in glioblastoma and 2) to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma.

BACKGROUND:

The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response.

METHODS:

Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry.

RESULTS:

MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001). However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297). The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing), and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis.

DISCUSSION AND CONCLUSION:

MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue.     

Anti-prothrombin antibodies are associated with adverse pregnancy outcome

Citation Marozio L, Curti A, Botta G, Canuto EM, Salton L, Tavella AM, Benedetto C. Anti‐prothrombin antibodies are associated with adverse pregnancy outcome. Am J Reprod Immunol 2011; 66: 404–409 Problem Women with antiphospholipid antibodies (aPL) such as lupus anticoagulant, anticardiolipin antibodies, and anti‐β₂ glycoprotein‐1 antibodies are at high risk of late pregnancy complications, such as severe pre‐eclampsia, placental insufficiency, and fetal loss. It has been observed that aPL consists of a heterogeneous group of antibodies targeting several phospholipid‐binding plasma proteins, including also anti‐prothrombin (anti‐PT), anti‐protein S (anti‐PS), and anti‐protein C (anti‐PC) antibodies. Their potential role in late pregnancy complications is not known. The aim of this work was to investigate the association between those autoantibodies and histories for adverse pregnancy outcome. Method of study Anti‐PT, anti‐PS, and anti‐PC antibodies were evaluated in 163 patients with previous severe pre‐eclampsia, fetal death, and/or placental abruption and in as many women with previous uneventful pregnancies, negative for aPL. Results The prevalence of anti‐PT antibodies was higher in cases than in controls (OR, 95% CI: 10.92, 4.52–26.38). The highest prevalence was observed in subjects with fetal death. Conclusion Anti‐PT antibodies appear to be associated with adverse pregnancy outcome, irrespectively of aPL.     

Differentially expressed genes in giant cell tumor of bone

Giant cells tumors of bone (GCTB) are benign in nature but cause osteolytic destruction with a number of particular characteristics. These tumors can have uncertain biological behavior often contain a significant proportion of highly multinucleated cells, and may show aggressive behavior. We have studied differential gene expression in GCTB that may give a better understanding of their physiopathology, and might be helpful in prognosis and treatment. Rapid subtractive hybridization (RaSH) was used to identify and measure novel genes that appear to be differentially expressed, including KTN1, NEB, ROCK1, and ZAK using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry in the samples of GCTBs compared to normal bone tissue. Normal bone was used in the methodology RaSH for comparison with the GCTB in identification of differentially expressed genes. Functional annotation indicated that these genes are involved in cellular processes related to their tumor phenotype. The differential expression of KTN1, ROCK1, and ZAK was independently confirmed by qRT-PCR and immunohistochemistry. The expression of the KTN1 and ROCK1 genes were increased in samples by qRT-PCR and immunohistochemistry, and ZAK had reduced expression. Since ZAK have CpG islands in their promoter region and low expression in tumor tissue, their methylation pattern was analyzed by MSP-PCR. The genes identified KTN1, ROCK1, and ZAK may be responsible for loss of cellular homeostasis in GCTB since they are responsible for various functions related to tumorigenesis such as cell migration, cytoskeletal organization, apoptosis, and cell cycle control and thus may contribute at some stage in the process of formation and development of GCTB.     

Computer assisted learning in manipulative therapy education

Teaching manipulative therapy is one of the most relevant issues in a physiotherapy course. However, for an effective instruction of this topic practical experience is considered fundamental. To achieve this purpose, this paper presents a computer assisted environment for the practical learning of manipulative therapy. The implementation of such a laboratory includes standard Personal Computers (PCs), the popular software package Matlab, a digital camera and gloves with embedded strength sensors located in the thumb and near the wrist. The students wear a glove that measures the applied force in the patient. These signals are?acquired and stored in a computer providing the teachers with the necessary information to analyze the?applied force and verify if the manual therapy was successful. A digital camera is also used to record the images of the student's movements during the manual therapy application. The developed system has an autonomy of 15h allowing the application of thirty therapies and the corresponding data analysis.     

Cytokeratin expression in central mucoepidermoid carcinoma and glandular odontogenic cyst

Central mucoepidermoid carcinoma (MEC) is an entity whose origin is still controversial. Glandular odontogenic cyst (GOC) is a recently described lesion whose relationship to low-grade central MEC has been reported in the literature. Our aim was to assess the cytokeratin (CK) profile of central MEC and GOC, and compare the results with CK expression in salivary gland MEC and odontogenic cysts and tumors. Eighty-five cases, including 6 central MECs, 23 salivary gland MECs, 10 GOCs, 34 odontogenic cysts and 12 ameloblastomas, were studied through immunohistochemistry using eleven monoclonal anti-CK antibodies. All central MECs expressed CKs 5, 7, 8, 14, and 18 and all GOCs expressed CKs 5, 7, 8, 13, 14, and 19. Comparing CK expression from GOC and central MEC we found differences in CKs 18 (30% vs 100%) and 19 (100% vs 50%). Central MEC and GOC are probably distinct entities with CK profiles similar to lesions of glandular and odontogenic origins, respectively, and expression of CKs 18 and 19 could be useful in their differential diagnosis.     

HIF-1α-independent hypoxia-induced rapid PTK6 stabilization is associated with increased motility and invasion

PTK6/Brk is a non-receptor tyrosine kinase overexpressed in cancer. Here we demonstrate that cytosolic PTK6 is rapidly and robustly induced in response to hypoxic conditions in a HIF-1-independent manner. Furthermore, a proportion of hypoxic PTK6 subsequently re-localized to the cell membrane. We observed that the rapid stabilization of PTK6 is associated with a decrease in PTK6 ubiquitylation and we have identified c-Cbl as a putative PTK6 E3 ligase in normoxia. The consequences of hypoxia-induced PTK6 stabilization and subcellular re-localization to the plasma membrane include increased cell motility and invasion, suggesting PTK6 targeting as a therapeutic approach to reduce hypoxia-regulated metastatic potential. This could have particular significance for breast cancer patients with triple negative disease.     

Synergy between electrical coupling and membrane properties promotes strong synchronization of neurons of the mesencephalic trigeminal nucleus

Electrical synapses are known to form networks of extensively coupled neurons in various regions of the mammalian brain. The mesencephalic trigeminal (MesV) nucleus, formed by the somata of primary afferents originating in jaw-closing muscles, constitutes one of the first examples supporting the presence of electrical synapses in the mammalian CNS; however, the properties, functional organization, and developmental emergence of electrical coupling within this structure remain unknown. By combining electrophysiological, tracer coupling, and immunochemical analysis in brain slices of rat and mouse, we found that coupling is mostly restricted to pairs or small clusters of MesV neurons. Electrical transmission is supported by connexin36 (Cx36)-containing gap junctions at somato-somatic contacts where only a small proportion of channels appear to be open (～0.1%). In marked contrast with most brain structures, coupling among MesV neurons increases with age, such that it is absent during early development and appears at postnatal day 8. Interestingly, the development of coupling parallels the development of intrinsic membrane properties responsible for repetitive firing in these neurons. We found that, acting together, sodium and potassium conductances enhance the transfer of signals with high-frequency content via electrical synapses, leading to strong spiking synchronization of the coupled neurons. Together, our data indicate that coupling in the MesV nucleus is restricted to mostly pairs of somata between which electrical transmission is supported by a surprisingly small fraction of the channels estimated to be present, and that coupling synergically interacts with specific membrane conductances to promote synchronization of these neurons.     

Functional dependence and caregiver burden in Alzheimer's disease: a controlled trial on the benefits of motor intervention

Background: Cognitive decline has a negative impact on functional activities in Alzheimer's disease. Investigating the effects of motor intervention with the intent to reduce the decline in functionality is an expected target for patients and caregivers. The aim of this study was to verify if a 6‐month motor intervention programme promoted functionality in Alzheimer's patients and attenuated caregivers' burden. Methods: The sample comprised 32 community patients with Alzheimer's disease and their 32 respective caregivers. Patients were divided into two groups: 16 participated in the motor intervention programme and 16 controls. Subjects performed 60 minutes of exercises, three times per week during the 6‐month period, to improve flexibility, strength, agility and balance. Caregivers followed the procedures with their patients during this period. Functionality was evaluated by the Berg Functional Balance Scale and the Functional Independence Measure. Caregivers completed the Neuropsychiatric Inventory Caregiver Distress Scale and the Zarit Carer Burden Scale. Two‐way ANOVA was used to verify the interaction between time (pre‐ and post‐intervention) and the motor intervention program. Results: While patients in the motor programme preserved their functionality, as assessed by the Functional Independence Measure, the controls suffered a relative decline (motor intervention group: from 109.6 to 108.4 vs controls: from 99.5 to 71.6; P= 0.01). Patients from motor intervention also had better scores than the controls on functional balance assessed by Berg scale (F: 22.2; P= 0.001). As assessed by the Neuropsychiatric Inventory and Zarit scale, burden was reduced among caregivers whose patients participated in the motor intervention programme compared with caregivers whose patients did not participate in this programme (Neuropsychiatric Inventory, caregiver's part: F: 9.37; P= 0.01; Zarit: F: 11.28; P= 0.01). Conclusion: Patients from the motor intervention group showed reduced functional decline compared to the controls, and there was an associated decrease in caregivers' burden.